Chaudry G J, Farrell K B, Ting Y T, Schmitz C, Lie S Y, Petropoulos C J, Eiden M V
Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Virol. 1999 Apr;73(4):2916-20. doi: 10.1128/JVI.73.4.2916-2920.1999.
The Chinese hamster cell lines E36 and CHOK1 dramatically differ in susceptibility to amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GALV); E36 cells are highly susceptible to both viruses, CHOK1 cells are not. We have previously shown that GALV can infect E36 cells by using both its own receptor, HaPit1, and the A-MuLV receptor, HaPit2. Given that the two cell lines are from the same species, the loss of function of both of these receptors in CHOK1 cells is surprising. Other studies have shown that CHOK1 cells secrete proteins that block A-MuLV entry into CHOK1 as well as E36, suggesting the two A-MuLV receptors are functionally identical. However, CHOK1 conditioned medium does not block GALV entry into E36, indicating the secreted inhibitors do not block HaPit1. HaPit1 and ChoPit1 therefore differ as receptors for GALV; ChoPit1 is either inactivated by secreted factors or intrinsically nonfunctional. To determine why GALV cannot infect CHOK1, we cloned and sequenced ChoPit1 and ChoPit2. ChoPit2 is almost identical to HaPit2, which explains why CHOK1 conditioned medium blocks A-MuLV entry via both receptors. Although ChoPit1 and HaPit1 are 91% identical, a notable difference is at position 550 in the fourth extracellular region, shown by several studies to be crucial for GALV infection. Pit1 and HaPit1 have aspartate at 550, whereas ChoPit1 has threonine at this position. We assessed the significance of this difference for GALV infection by replacing the aspartate 550 in Pit1 with threonine. This single substitution rendered Pit1 nonfunctional for GALV and suggests that threonine at 550 inactivates ChoPit1 as a GALV receptor. Whether native ChoPit1 functions for GALV was determined by interference assays using Lec8, a glycosylation-deficient derivative of CHOK1 that is susceptible to both viruses and that has the same receptors as CHOK1. Unlike with E36, GALV and A-MuLV exhibited reciprocal interference when infecting Lec8, suggesting that they use the same receptor. We conclude both viruses can use ChoPit2 in the absence of the inhibitors secreted by CHOK1 and ChoPit1 is nonfunctional.
中国仓鼠细胞系E36和CHOK1对嗜双性小鼠白血病病毒（A-MuLV）和长臂猿白血病病毒（GALV）的易感性存在显著差异；E36细胞对这两种病毒都高度易感，而CHOK1细胞则不易感。我们之前已经表明，GALV可以通过自身受体HaPit1和A-MuLV受体HaPit2感染E36细胞。鉴于这两种细胞系来自同一物种，CHOK1细胞中这两种受体功能的丧失令人惊讶。其他研究表明，CHOK1细胞分泌的蛋白质会阻止A-MuLV进入CHOK1细胞以及E36细胞，这表明两种A-MuLV受体在功能上是相同的。然而，CHOK1条件培养基并不能阻止GALV进入E36细胞，这表明分泌的抑制剂不会阻断HaPit1。因此，HaPit1和ChoPit1作为GALV的受体存在差异；ChoPit1要么被分泌因子失活，要么本身就无功能。为了确定GALV为何不能感染CHOK1细胞，我们对ChoPit1和ChoPit2进行了克隆和测序。ChoPit2与HaPit2几乎相同，这就解释了为什么CHOK1条件培养基能通过两种受体阻断A-MuLV的进入。尽管ChoPit1和HaPit1有91%的同源性，但一个显著差异出现在第四个细胞外区域的第550位，多项研究表明该位置对GALV感染至关重要。Pit1和HaPit1在第550位是天冬氨酸，而ChoPit1在该位置是苏氨酸。我们通过将Pit1第550位的天冬氨酸替换为苏氨酸来评估这种差异对GALV感染的意义。这一单一位点的替换使Pit1对GALV失去功能，这表明第550位的苏氨酸使ChoPit1作为GALV受体失活。通过使用Lec8进行干扰试验来确定天然ChoPit1是否对GALV起作用，Lec8是CHOK1的糖基化缺陷衍生物，对这两种病毒都易感，并且与CHOK1具有相同的受体。与E36不同，GALV和A-MuLV在感染Lec8时表现出相互干扰，这表明它们使用相同的受体。我们得出结论，在没有CHOK1分泌的抑制剂的情况下，两种病毒都可以使用ChoPit2，并且ChoPit1无功能。
